SirT1 is required in the male germ cell for differentiation and fecundity in mice

Abstract

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

Keywords: Male germ cell; Reproduction; SirT1.

Fig. 1

. SirT1 is highly expressed in the testis. (A) Immunoblot of SirT1 in mouse tissues. Each lane contains 50 µg of protein. (B) Immunofluorescence of SirT1 protein in cross sections of seminiferous tubules from wild-type and SirT1 whole body KO mice. (C) Immunohistochemistry for SirT1 counterstained with PAS/H in the XII stages of the seminiferous tubules. Representative images of stages I-XII taken with a 100× objective. The table on the right indicates the relative abundance in the different cell types, ± indicates that some of these cell populations demonstrate light staining. BAT, brown adipose tissue; WAT, white adipose tissue; Spg_A, A type spermatogonia; Spg_In, Intermediate spermatogonia; Spg_B, B type spermatogonia; Pl, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; M, meiotic; S, spermatid.

Fig. 2.

Deletion of SirT1 in pre-meiotic cells decreases testis weight and sperm number. (A) SirT1 immunoblotting on whole testis lysate from three biological replicates of 3-month-old SirT1f/f (WT) and Stra8-iCre; SirT1f/f (KO) mice. (B) Testis weight per gram of body weight in SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. Images are of 12-week-old WT and KO mice. (C) Number of sperm per caudal epididymis from 12-week-old SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. The line represents the mean sperm number. (D) PAS/H-stained cross sections of seminiferous tubules from 5-week- and 12-week-old SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. Black arrows indicate elongating and/or condensing spermatids, and arrowheads indicate dying and/or dead cells. (E) The number of TUNEL-positive cells per tubule cross section in SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice aged 5 weeks or 12 weeks. (F) Pachytene spermatocytes are identified by the γH2AX positive sex body (top; arrow). Quantification of tubules that contain pachytene spermatocytes in SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) 12 days post-partum mice (bottom). n=3 mice per group. Scale bars: 50 µm (D); 20 µm (F).

Fig. 3.

Post-translational modifications of histones are altered in Stra8-Cre KO testes. (A) Pan-acetyl lysine (AcK) immunoblot of acid soluble proteins from the chromatin of germ cells isolated from three littermate pairs of 3-month-old SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. Coomassie Blue staining is shown at the bottom. (B) Quantification of iTRAQ abundance for histone H4 acetylated tryptic peptides from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) whole testes, n=3. (C) Immunoblots of histone H4 acetylation on acid soluble proteins isolated from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) germ cell chromatin. (D) Acetylated histone H4K5 (H4Ack5) levels in the non-chromatin NETN soluble fraction from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) germ cells. (E) Immunoblots of acid soluble proteins from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) germ cell chromatin for histone H3 and H2B post-translational modifications. me2, di-methylated, me3, tri-methylated. (F) Immunoblots of H4, H3, and H2B acetylation on acid extracts of SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) germ cell chromatin from mice of different ages (days postpartum). (G) Immunofluorescence in stage IX-X tubules from 12-week-old SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice for H4AcK5. Quantification of fluorescence intensity of images (n=9 total sections from three littermate pairs; three stage IX-X tubules per pair of mice). Scale bar: 50 µm.

Fig. 4.

The histone to protamine transition is altered in Stra8-Cre KO testes. (A) Squash slides from stage IX-XII seminiferous tubules stained for transition protein 2 (TP2) and (B) protamine 1 (Prm1). Images are representative of multiple squash slides prepared from three sibling pairs of SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. (C) Immunoblot of histone H4 from sonication-resistant spermatid (SRS) lysates isolated from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. (D) Testis-specific histone H2B (TH2B) immunofluorescence on caudal epididymal cross sections (left) and quantification of sperm with staining of TH2B per nucleus (DAPI, blue) (right, n=4 mice; one section per mouse). (E) Immunoblot of TH2B from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) sperm lysates. Scale bars: 10 µm (A,B); 50 μm (D).

Fig. 5.

Pre-meiotic SirT1 KO mice have abnormal spermatozoa and decreased chromatin condensation in elongating spermatids. (A) Electron micrograph images of elongating spermatids from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice. (B) Quantification of relative electron density from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice, N=161 and 119 respectively. Insets in A and B show enlarged images of sections. (C) Phase-contrast images of normal and abnormal sperm (left) and the quantification of normal and abnormal sperm from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice (right). A minimum of 200 spermatozoa were counted. (D) Quantification of a comet assay on mature spermatozoa from SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) animals. Per mouse, 200 sperm cells were scored. Line represents mean number of comet tail-positive cells.

Fig. 6.

SirT1 is necessary to maintain male fecundity with age. (A-D) SirT1 f/f (WT) and Stra8-Cre; SirT1 f/f (KO) mice were mated to WT females and the number of pups per litter (A), total number of litters (B), cumulative number of pups (C) and mean age to reproductive senescence (D) were determined (n=10). (E) SirT1 protein levels, using immunoblotting, in the testes of mice aged 3, 16, or 33 months old.